This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The DNA in every cell is packaged and folded several times to fit into a small organelle, the nucleus. The three-dimensional (3D) packaging of DNA can influence gene activity by positioning of genes near nuclear compartments, small regions in the nucleus with distinct sets of proteins that promote certain biochemical activities. The nuclear periphery is one such compartment. Although the mechanism that targets genes to the nuclear periphery is likely to play an important role in gene regulation, how genes are targeted to this compartment is unknown. Hence, the goal of this study is to identify sequences that position genes at the nuclear periphery. We have identified a region on mouse chromosome 14 that localizes to the nuclear periphery of embryonic stem (ES) cells. This region contains gene-depleted sequences, gene "deserts", that are positioned closer to the nuclear edge than are the neighboring genes. This study tests the hypothesis that specific gene deserts sequences actively target chromatin to the nuclear periphery. This hypothesis will be tested by (1) identifying the sequences within gene deserts that most frequently associate with the nuclear periphery, and (2) testing desert sequences for a direct role in targeting chromatin to this compartment by inserting these sequences into other positions in the mouse genome. These aims will be accomplished using state-of-the-art techniques to investigate nuclear structure, including fluorescence in situ hybridization (FISH) of large regions of chromosomes, high-resolution 3D microscopy to precisely map gene positions, and genome engineering in mouse ES cells.